The aim of the project is to characterize the interactions of the formyl peptide ligands with formyl peptide receptors and to use these peptides as models for: 1) developing the rapid mix flow cytometer and 2) studying signal transduction. A family of formyl peptides with fluorescein isothiocyanate conjugated at positions 2 through 7 has been generated and their binding to receptors has been characterized by flow cytometry with respect to binding and cell physiology (Vilven and Freer, manuscript in press). These studies have suggested that the family of peptides may be used in the future to detect sites of contact by photoaffinity crosslinking along with capillary electrophoresis and sensitive detection. Prossnitz (who has now moved to New Mexico) and Ye have produced mutant receptors whose kinetics are being characterized in the rapid mix flow cytometer. The molecular assemblies, receptor processing, and desensitization of these receptors are being evaluated. The data suggest that receptors exist in two new forms: the native form which is different from that detected in cell membrane preparations, and a receptor form which is not yet phosporylated but is nonetheless beginning to be regulated in the signal transduction pathway. The organization of these receptors in the membrane will be evaluated by fluorescence energy transfer in flow cytometry. The next phase of the project will involve a description of the underlying biochemistry of signal transduction. The kinetic methods will combine rapid mix flow cytometry with biochemisty to evaluate the evolution of receptors among different macromolecular complexes. Transduction complexes are being assembled on beads in both solublized and membrane bound forms to examine the steps in signal transduction. We have met with Neubig and his student on two occasions during year 16 to develop experimental strategies.